Part:BBa_K5443002:Experience

Applications of BBa_K5443002

We showed that the Sam5 C3H enzyme was functional in E. coli as part of a vanillin biosynthetic pathway, as evidenced by production of caffeate (see LC-MS data from one clone pMQ3C-11, Fig. 1). Further evidence for functional expression of Sam5 was seen from production of a brown melanin-type dye as a side-reaction (see Fig. 2); we believe this indicates that Sam5 is acting on tyrosine (to make L-DOPA, which polymerises) in addition to our intended reaction on p-coumarate to make caffeate.

Figure 1. LCMS analysis of compounds produced using pMQ3C-11.
The graph shows the detected presence of all 6 tested compounds, validating successful expression of all parts within pMQ3C-11, including Sam5.

Figure 2. Patched clones of plasmid variants.
Each plate shows patched colonies transformed with variants of our created plasmids pMQ3B and pMQ3C. Some patched colonies show diffusion of a brown pigment into the surrounding agar, suggesting a side-reaction of the enzyme Sam5 that produces the brown pigment L-DOPA.

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